The present invention comprises a process enabling simultaneous detection of two transgenes using a Multiplex Polymerase Chain Reaction (MPCR), particularly the detection of human serum albumin (HS A) gene (responsible for production of human blood serum protein as a bio-pharmaceutical product) and bar (phosphinothricin acetyltransferase) gene that is responsible for is phosphinothricin tolerance in transgenic wheat. The invention can be successfully employed in rapid and simultaneous detection of these two transgenes with high reproducibility and sensitivity in transgenic wheat.
Prior Arts
Studies for detection of an exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and an endogenous lectin gene in genetically modified soya foodstuff and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and CaMV 35S promoter in transgenic soybean has been undertaken.
Example 1:
Hurst CD, A Knight and IJ Bruce 1999, PCR detection of genetically modified soya and maize in foodstuffs. Molecular Breeding. Vol. 5: 579-586, detected an exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and an endogenous lectin gene in transgenic soybean simultaneously. DNA was extracted from the transgenic soybean food-stuff. Primers for both the genes were designed and synthesized. The primer pair for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene is forward primer of 25 base pair (5'-CCACTATCCTTCGCAAGACCCTTCC-3') and reverse primer of 24 base pair (5'-CTTCTGTGCTGTAGCCACTGATGC-3'). The primer pair for lectin gene is forward primer of 22 base pair (5'-GAAGCAACCAAACATGATCCTC-3') and reverse primer of 24 base pair (5'-ATGGATCTGATAGAATTGACGTTA-3'). The 25µl polymerase chain reaction (PCR) reaction contained 5µl of the extracted DNA, 0.2µM of each primer for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and 0.6uM of each primer for lectin gene, 1.5 units of Taq DNA polymerase, lOmM Tris HC1 (pH 9 at RT), 50mM KC1, 1.5mM MgCl2, 200µM of each dNTP and BSA. Three-temperature multiplex polymerase chain reaction for detection of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and lectin gene was standardized using GeneAmp PCR System 2400 (Perkin Elmer, UK). Multiplex polymerase chain reaction
performed with 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) F/R and lectin F/R primers involved an initial denaturation at 95°C for 5 min followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec, 72°C for 60 sec and finally reaction was held at 72°C for 5 min. Primers amplified 320 base pair fragment for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and 407 base pair of lectin gene (Figure 1 on drawing sheet 1).
Example 2:
P. K. Firke and Gurinder Jit Randhawa 2005a, Polymerase Chain Reaction for evaluation /detection of transgenic planting material. Indian J. Plant Genet. Resour 18 (1) 143, detected 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and CaMV 35S promoter separately using traditional Polymerase Chain Reaction (PCR). DNA was extracted from crushed seeds of transgenic soybean using protocol of Dellaporta et al. (1983) with minor modifications in the purification steps. The extracted DNA was purified and quantified on fluorimeter (Hoefer) using DNA binding fluorescent Hoechst-33258 dye. The primer pair was designed for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene using Primer Select Module of Laser Gene DNA Star Software whereas the primer pair for CaMV 35S promoter used was designed by Pietsch K et al. 1997, Screeningverfahren zur identifizierung gentechnisch veranderter lebensmittel, Deut Lebensm Rundschau Heft, 2, 35-38. The primer pair for amplifying EPSPS gene comprising of forward primer of 20 base pair (5'-AACGGTGGACTCCTTGCTCC-3'), and reverse primer of 21 base pair (5'-GGTAGCACCTTCAGCGAATGC-3') and the primer pair for amplifying CaMV 35S promoter comprising of forward primer of 19 base pair (5'-GCTCCTACAAATGCCATCA-3'), and reverse primer of 20 base pair (5'-GATAGTGGGATTGTGCGTCA-3') were synthesized by Sigma Genosys (USA). The conditions to generate reproducible amplified DNA fragments were optimized by varying the components of PCR reaction mixture and the temperature regimes. In a final volume of 25 uL, the optimized amplification reaction contained 25 ng genomic DNA, lx assay buffer (10 mM Tris-Cl pH 8, 1.5 mM MgCl2, 50 mM KC1 and 0.01 gelatin), 0.2 mM dNTPs, 10 pmol of each primer and 1 Unit of Taq DNA polymerase (MBI Fermentas, USA). Best amplification of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene was observed when the PCR programme involved an initial denaturation at 95°C for 3 min followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec, 72°C for 60 sec and finally reaction was held at 72°C for 3 min. Primer
pair amplified 320bp fragment of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene sequence (Figure 2 on sheet 2).
Best amplification of CaMV 35S promoter was observed when the PCR programme involved an initial denaturation at 95 C for 3 min followed by 40 cycles of 94°C for 20 sec, 54 C for 40 sec, 72°C for 60 sec and finally reaction was held at 72°C for 5 min. Primer pair amplified 195bp fragment of CaMV 35S promoter sequence (Figure 3 on sheet 3).
Example 3:
Firke PK and GJ Randhawa, 2005b, Polymerase chain reaction for testing of transgenic planting material, a presentation was made in "International Conference on Plasticulture and Precision Farming, November 17-21, New Delhi, pp 315. An exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and CaMV 35S promoter gene were detected simultaneously in transgenic soybean. DNA was extracted from the crushed seeds of transgenic soybean. Primers for both the genes were designed and synthesized. The 25µl polymerase chain reaction (PCR) reaction contained 5µl of the extracted DNA, 0.5µM of each primer for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and 0.5µM of each primer for CaMV35S promoter, 1 Unit of Taq DNA polymerase, lOmM Tris HC1 (pH 9 at RT), 50mM KC1, 1.5mM MgCl2,200µM of each dNTPs.
Three-temperature multiplex polymerase chain reaction for detection of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and CaMV35S promoter was standardized using PE-9600 Thermal cycler. Multiplex polymerase chain reaction performed with EPSPS-F/R and CaMV 35S-F/R primers involved an initial denaturation at 95°C for 5 min followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec, 72°C for 60 sec and finally reaction was held at 72°C for 5 min. A fragment of 441 base pair was amplified for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and fragment 131 base pair of CaMV 35S promoter was amplified (Figure 4 on drawing sheet 4).
Disadvantages of the Prior Arts:
The simultaneous detection of 5 -enolpyruvylshikimate-3 -phosphate synthase (EPSPS) gene and endogenous lectin gene by Hurst 1999 was carried out in food-stuff whereas in the present invention both the genes detected are exogenous and are detected from the planting material which is of immediate relevance for detection of transgenic from non transgenic for bulk imports. Detection of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and CaMV 35S promoter separately by Firke and Gurinder Jit Randhawa (2005a) by traditional PCR detect only one transgene at a time which reduces the efficiency of detection system and can sometimes detect false positives or false negatives as well. Also, the simultaneous detection of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and CaMV 35S promoter by Firke and Gurinder Jit Randhawa 2005b by multiplex PCR in transgenic soybean. Yet, there is not even a single report available to detect human serum albumin gene and bar gene simultaneously in transgenic wheat or any other transgenic crop plant.
Wheat is the most widely grown cereal crop globally and is major staple diet of billions of people in developing countries. Genetic engineering is of significant interest to improve productivity by overcoming biotic and abiotic stress and nutritional enhancement. Recent progress in transformation methods has made it possible to transfer and express genes from other species into wheat to achieve agronomically desirable traits, such as herbicide tolerance (Pellegrineschi et al. 2002, Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants, Genome 45: 421-430). Among the food crops, wheat is one of the most abundant source of energy for the world population especially for the poor people of the developing world. The genetic improvement of wheat has received considerable attention over the last few years from plant breeders with the purpose of increasing the grain yield, to minimize crop loss due to unfavorable environmental conditions, and attack by various pests and pathogens.
'Bobwhite' SH 98 26 was identified as a super-transformable wheat line by A. Pellegrineschi et al. (2002), Genome 45(2): 421-430. Furthermore, with the wide spread introduction of new transgenic crops, detection of transgenes will become even more complex. Even though traditional polymerase chain reaction (PCR) for detecting transgenic crops has
fairly high sensitivity and specificity, only one target gene can be detected whereas multiplex polymerase chain reaction has the same sensitivity and specificity but is more efficient and economical as compared to traditional polymerase chain reaction (PCR) and can be employed for detecting more than one target transgenes.
Novelty of the present Invention:
The process comprising the present invention is novel as the sequences of the primer pairs used for the amplification of the human serum albumin gene and bar gene, the polymerase chain reaction (PCR) protocol /method employed have not been used earlier. This is the first report of the simultaneous detection of human serum albumin gene and bar gene using the combination of the novel primer sequences and multiplex polymerase chain reaction (PCR) method in transgenic wheat.
Inventive step of the present Invention:
India is at the threshold stage of developing and importing transgenic crops. With the widespread introduction and commercialization of transgenic crops the requirement to detect transgenes in transgenic crops with maximum efficiency is becoming all the more important. Multiplex polymerase chain reaction used in the present invention is more efficient and reliable as compared to traditional polymerase chain reaction (PCR) and can be employed for detecting more than one target transgenes with almost similar resources (except the primers).
Industrial aspect of the present Invention:
The transgenic wheat for human serum protein (albumin) production as biopharmaceutical product and phosphinothricin herbicide tolerance targeted in this invention has been imported in India during year 2004 due to which it has become all the more important to develop reliable, efficient and precise protocols for detection of the transgenes i.e. human serum albumin (HAS) and bar genes incorporated in it to meet the regulatory requirement and to solve the legal disputes in future as and when these arise.
The five major transgenic crops maize, soybean, cotton, canola and potato are growing in commercially large areas in different countries. (C. James, 2005, Preview: Global status of commercialized biotech/GM crops:. ISAAA Briefs). Other major food crops on which lot of research work is going on to improve their nutritional quality and other traits include rice and wheat. Wheat being the staple food of billions of people in developing world has been target for genetic improvement since last decade.
The present invention therefore holds immense commercial potential as with the large-scale future imports in the transgenic wheat, efficient and economic detection kits would be a necessity to meet the regulatory requirements and for solving legal dispute and if they arise.
The main objective of the present invention is to develop a protocol for the rapid detection of transgenes in transgenic wheat and to develop a polymerase chain reaction (PCR) programme for simultaneous detection of more than one transgenes in transgenic wheat.
As such, this invention comprises an innovative process for simultaneous detection of two transgenes using a multiplex polymerase chain reaction, where in human serum albumin and bar genes have been detected in transgenic wheat using a combination of novel primers and polymerase chain reaction (PCR) programme.
Advantages of the present invention:
i) Process developed in the present invention is much more economical, rapid and precise as compared to the traditional polymerase chain reaction (PCR) as more than one transgenes can be detected in a single reaction with almost similar resources (except the primer).
ii) The process is much more efficient than the traditional polymerase chain reaction (PCR).
iii) Bar gene is used as the most common selectable marker to transform most of the currently approved transgenic crops. The use of the bar gene allows the detection of most of the approved transgenic crops. Multiplex polymerase chain reaction process can confirm whether wheat is genetically modified as we can detect human serum albumin gene along with bar gene in a single experiment, thus the process is much more efficient and precise.
4. DETAILED DESCRIPTION OF THE PRESENT INVENTION:
DNA was extracted from the crushed seeds of transgenic wheat contining human serum (HAS) and bar gene as per the method of Dellaporta SL, J Wood, and JB Hicks (1983, A plant DNA mini-preparation: version II. Plant Molecular Biology Reporter. Vol. 19: 11-15.) The concentration of the DNA in the sample was determined with Hoefer DNA fluorimeter model DQ200 (Hoefer Pharmacia Biotech inc. San Fransisco, CA) using Hoechst 33258 as the dye and calf thymus DNA as the standard. DNA samples were diluted in 10:1 (Tris HC1: Ethylene Diamine Tetra Acetic acid) to a working concentration of approximately 5ng/µl and stored at 4°C until polymerase chain reaction (PCR) amplification.
Primers were designed using Primer Select Module of the Laser Gene Software (DNA Star Inc, USA) and were synthesized by Genuine Chemical Corporation, New Delhi, India. The primer pair designed for human serum albumin (HAS) gene is
HSA-F Forward 5'-TCCTCATCAGCGCCTCAGTTATCA-3'
HSA-R Reverse 5'-CACAGCAGTCAGCCATTTCACCAT-3'
The primer pair for bar gene is
BAR-F Forward 5'-CCGTACCGAGCCGCAGGAACC-3'
B AR-R Reverse 5' -GGC AGCCCGATG AC AGCGACC AC-3'
Three temperatures multiplex polymerase chain reaction (PCR) for detection of human serum albumin (HAS) gene and bar gene was standardized on PTC-200 Thermal cycler (MJ Research, Inc.). The 15µl multiplex polymerase chain reaction mixture volume consisted of 15ng of genomic DNA, lx reaction buffer (10 mM tris HC1, pH 8.3 and 50 raM KC1), 1.5mM MgCl2, 200uM of each dNTPs, 0.6 Unit of Taq DNA polymerase (all reagents from MBI Fermentas, USA) and 5µM of Forward and Reverse primer for human serum albumin (HAS) gene and Forward and Reverse primer of bar gene. 100 base pair DNA ladder (MBI Fermentas, USA) was used to confirm the size of the amplified product.
For the amplification of the transgene sequences different temperature regimes were used in three-temperature polymerase chain reaction (PCR) cycle. The multiplex polymerase chain reaction method involved an initial denaturation at 93 - 97°C for 4 - 10 min followed by 40 - 50 cycles of 94 - 95°C for 20 - 90 sec, 55 - 65°C for 20-120 sec, 72 - 73°C for 60-120 sec and finally reaction was held at 72°C for 3-7min.
Best amplification of human serum albumin (HAS) gene and bar gene was observed when the multiplex polymerase chain reaction programme involved an initial denaturation at 94°C for 5 min followed by 45 cycles of 94°C for 35 sec, 60°C for 70 sec, 72°C for 60 sec and finally reaction was held at 72°C for 7 min. The amplification products were electrophoresed on 3% agarose gel run on horizontal gel electrophoresis system stained with ethidium bromide. Amplification products of 311 base pair for human serum albumin (HAS) gene sequence and 279 base pair fragment for bar gene were detected using primer pairs HAS-F/R and BAR-F/R (Figure 5 on drawing sheet 5).
The routine polymerase chain reaction (PCR) involves only single primer pair for amplification of single gene whereas multiplex polymerase chain reaction has the same sensitivity and specificity but it is more efficient than routine polymerase chain reaction (PCR) because in a single multiplex polymerase chain reaction (PCR) reaction where more than one primer pair can be used to amplify more than one specific transgenes. Thus the multiplex polymerase chain reaction in the present invention is much more economical, rapid and precise than routine polymerase chain reaction (PCR).

WE CLAIM:
1. Process enabling simultaneous detection of transgenes namely
human serum albumin (HAS) and bar genes in transgenic wheat
comprises:
(Sequance Removed)
Wherein primer pair of seq. ID 1 amplifies 311 base pairs of HAS gene and seq. ID 1 amplifies 279 base pairs of bar gene respectively and the MPCR method comprising of initial denaturation at 94°C for 5 min followed by 45 cycles of 94°C for 35 sec, 60°C for 70 sec, 72°C for 60 sec, finally reaction was held at 72°C for 7 min, the amplified products are electrophoresed on 3% agarose gel run on horizontal gel electrophoresis system followed by staining with ethidium bromide.
2. The process as claimed in claim 1, wherein PCR reaction mixture
comprises of 15µl consisting of 15ng of genomic DNA, IX reaction
buffer (10mM tris HC1, pH 8.3 and 50mM KC1), 1.5 mM Mgcl2,
200uM of each dNTPs, 0.6 Unit of Taq DNA polymerase and 5µM of
each primer pair.
3. A process for simultaneous detection of two transgenes human serum albumin (HAS) and bar gene in transgenic wheat utilizing a combination of novel primer pair of sequence ID 1 for amplifying HAS gene and of sequence ID 2 for amplification of bar gene in Multiplex Polymerase Chain Reaction (MPCR) using the PCR reaction mixture substantially as herein described along with the drawing.